A major effort is underway to define host complexes co-opted by HIV in order to develop next generation anti- retroviral therapies that are less susceptible to escape mutations. A promising drug target is the HIV accessory protein Vif, which coopts a host ubiquitin E3 ligase complex consisting Cullin-Ring Ligase 5 (CRL5) and core binding factor beta (CBF) to downregulate the APOBEC3 family of restriction factors. We have reconstituted the Vif-E3 ubiquitin ligase activity from recombinant components and propose to develop a series of primary and secondary screens to discover Vif-E3 ligase inhibitors. In Aim 1 we will develop an activity-based high- throughput screen for inhibitors of the Vif-E3 ligase. This screen eliminates potential bias in querying any given pair of protein interactions in the complex, maximizing the chance for inhibitor discovery. In Aim 2 we will develop a suite of secondary assays for following up on hits, including an orthogonal secondary high- throughput assay, a counter screen to determine selectivity of hits using a related cellular E3, biophysical assays such as SPR and NMR to report on target binding, mechanism of action studies, and assays to evaluate effects on viral infectivity. Unlike previous screens for Vif inhibitors, which were cell based, our approach employs recombinant components, allowing the discovery of inhibitors that target a defined biochemical system. This will facilitate target identification and lead optimizatin using the structure-based drug design paradigm. These screens could be applied to other viral accessory proteins that make use of the CRL machinery to downregulate restriction factors. Hits discovered in these screens could provide promising leads for next generation anti-retroviral therapies. Alternatively, they may provide molecular probes to explore diverse interactions between Vif and A3 family members or the role of CRL5 in auto-immune disease.